Could a blood test for PTSD and depression be on the horizon?

Could a blood test for PTSD and depression be on the horizon?

Article  in  Expert Review of Proteomics · November 2018

DOI: 10.1080/14789450.2018.1544894

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Could a blood test for PTSD and depression be on the horizon?

Dario Aspesi & Graziano Pinna

To cite this article: Dario Aspesi & Graziano Pinna (2018) Could a blood test for PTSD and depression be on the horizon?, Expert Review of Proteomics, 15:12, 983-1006, DOI:

10.1080/14789450.2018.1544894

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Accepted author version posted online: 05 Nov 2018.

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Could a blood test for PTSD and depression be on the horizon?

Dario Aspesi ^# and Graziano Pinna

The Psychiatric Institute, Department of Psychiatry, University of Illinois at Chicago, Chicago, IL, USA

ABSTRACT

Introduction: Depression and posttraumatic stress disorder (PTSD) are two complex and debilitating psychiatric disorders that result in poor life and destructive behaviors against self and others. Currently, diagnosis is based on subjective rather than objective determinations leading to misdiagnose and ineffective treatments. Advances in novel neurobiological methods have allowed assessment of promising biomarkers to diagnose depression and PTSD, which offers a new means of appropriately treating patients.

Areas covered: Biomarkers discovery in blood represents a fundamental tool to predict, diagnose, and monitor treatment efficacy in depression and PTSD. The potential role of altered HPA axis, epigenetics, NPY, BDNF, neurosteroid biosynthesis, the endocannabinoid system, and their function as biomarkers for mood disorders is discussed. Insofar, we propose the identification of a biomarker axis to univocally identify and discriminate disorders with large comorbidity and symptoms overlap, so as to provide a base of support for development of targeted treatments. We also weigh in on the feasibility of a future blood test for early diagnosis.

Expert commentary: Potential biomarkers have already been assessed in patients ’ blood and need to be further validated through multisite large clinical trial stratification. Another challenge is to assess the relation among several interdependent biomarkers to form an axis that identifies a specific disorder and secures the best-individualized treatment. The future of blood-based tests for PTSD and depression is not only on the horizon but, possibly, already around the corner.

ARTICLE HISTORY Received 1 May 2018 Accepted 2 November 2018 KEYWORDS

Major depressive disorder;

posttraumatic stress disorder; biomarker axis;

neurosteroids;

endocannabinoids;

diagnostic; blood test

1. Introduction

Coping with stress and negative life experience is an essential survival function not only in humans, but also in all living organisms. Environmental factors affect behavioral regulation and the ability to adapt to changed environmental conditions or the lack thereof, could play a key role in the onset of mood disorders. While adversities are very frequent during the life- time of an individual, ranging from 50% to 84% in the general population [1,2], the majority of people are resilient to adverse life events [3]. Nonetheless, a large portion of them (~10%) fails to develop resilience, and develops, instead, mood dis- orders, such as major depressive disorder (MDD) and/or post- traumatic stress disorder (PTSD) [4].

MDD and PTSD have been dramatically increasing in the past decade [5]. Depression is a profound multifaceted neuro- biological disorder of mood and emotions, which is often the result of different psychological stressors, has a prevalence of 8 –12% [ 6], and is the cause of impairment in different neu- ropsychological functions, like attention, learning, and mem- ory [7]. The DSM-5 describes a multitude of symptoms for MDD, such as sadness, anhedonia, disturbed concentration, significant changes in body weight (loss or gain) that highlight the complexity of this neuropsychopathology [8]. Likewise, PTSD is a stress-induced psychiatric disorder that emerges in

individuals after the exposure to a trauma and is characterized by an altered ability to cope with stress. The core symptoms of this psychiatric disorder are reexperiencing symptoms, night- mares about the trauma, changes in arousal and reactivity, avoidance of trauma-related reminders and alterations of mood [8]. PTSD and MDD share numerous overlapping symp- toms and comorbidity and often MDD symptoms in PTSD patients are a progression of the disorder [9]. Both PTSD and MDD are characterized by high incidence of suicidality with a prevalence of 9.5% of patients with MDD that attempted suicide over an 18-month period [10]. Predictors for suicide in MDD patients are the male gender, family history of psychia- tric disorders, more serious depressive symptoms and comor- bidity with other disorders, such as anxiety spectrum disorders and substance use disorder [11]. The core and the overlapping symptoms of MDD and PTSD are presented in Figure 1.

Moreover, both these neuropathologies show a gender- related dimorphism with females more affected than males [12,13]. In fact, in both PTSD and MDD, the prevalence in women is more than double that observed in males [14] pointing to a role of sexual hormones in the development and mainte- nance of the disorder. While, imbalance in sexual hormones synthesis could play an important role, they cannot explain the basic psychobiological mechanisms. Recently, it has become clearer that the convergence of multiple factors, such as

CONTACT Graziano Pinna [email protected]; [email protected] The Psychiatric Institute, Department of Psychiatry, University of Illinois at Chicago, 1601 W. Taylor Str., Chicago, IL 60612, USA

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Dario Aspesi is a PhD candidate who wrote the initial review draft.

https://doi.org/10.1080/14789450.2018.1544894

© 2018 Informa UK Limited, trading as Taylor & Francis Group

biological aspects and sociopsychological environmental condi- tions, play a role in MDD and PTSD [15 – 18]. Likewise, a multiplicity of factors, including genetic vulnerability, immuno- logical alterations, epigenetic mechanisms, neurohormones, neu- rotransmitter systems, neuropeptides and endocannabinoids and their receptors, seem to play a role in both the manifestation and the maintenance of mood disorders.

Despite advances in the neurobiological and pathophysio- logical aspects, the current diagnosis for MDD and PTSD is based on patients ’ self-reported interviews and on the clini- cian ’s observation. These methods to diagnose psychiatric disorders are quite subjective and far from optimal because of the consistent heterogeneity not only of the symptoms, but also of the disorder etiology [19]. Furthermore, patients with mood disorders frequently are not able to adequately charac- terize and describe the symptoms of the pathology [20] and the scoring systems are often discordant [21]. This scenario underscores the need to develop objective biological diagnos- tic tools based on neurobiological deficits to obtain early diagnosis, monitor individuals at risk, instruct individualized treatments, follow-up on treatment success and possibly pre- vent future relapse [22,23]. We anticipate that this will bring a disruption in the diagnosis of psychiatric disorders as we know of, based on symptoms characterized by the DSM-5 to regroup disorders based on their neurobiological characteris- tics. The progress in the diagnosis and treatment of psychiatric disorders based on biomarker assessment, rather than by

symptoms classification, is long needed. Contrary to other neuroscience fields, such as the neurodegenerative disorders, progress in the assessment of valid biomarkers in psychiatry has been rather slow [24].

In this review, we will summarize several promising biomar- kers in PTSD and MDD and weigh in on whether these offer hope in the development of a future blood test for these conditions. Tests will provide a useful tool for treatment selec- tion and to monitor treatment outcomes; ensuring patients receive the most appropriate and efficient treatment for a better chance to promptly recover.

1.1. Mood disorders and biomarker discovery

The identification of biomarkers in clinical psychiatry may give a measurable indicator of the neurobiological individual con- dition independently of a DSM-5-based diagnosis, of its pre- disposition to develop psychiatric disorders or the current presence of a behavioral dysfunction.

Biomarkers are objective measures of physiological and patho- logical processes or biological responses to a therapeutic interven- tion [25]. Biomarkers may include a gene or a set of genes, morphological characteristics, proteins, neuropeptides, or other biomolecules that are specifically altered in a disorder or disease [25]. Molecules identified as potential biomarkers allow accurate diagnosis and prognosis, to understand the pathogenesis and the pathophysiological mechanisms of the disorder, to predict disease

Figure 1. Core symptoms of MDD and PTSD described by DSM-5.

The DSM-5 describes the criteria to diagnose psychiatric disorders such as MDD and PTSD. The light blue circle lists the core symptoms of MDD, while, the orange circle lists the core

symptoms of PTSD. The overlapping area represents the shared symptoms of these two disorders.

progression, and to monitor the therapy progress. Biomarkers can also be useful to identify drug targets for the development of new therapeutic strategies [26].

Diagnostic tests are an urgent goal in the development of biomarkers for psychiatric disorders, because these tests will provide an objective approach to distinguish not only healthy individual from whom is affected by the disorder, but also can help classify correctly the subpopulations of patients with or without comorbidities. The use of imaging together with new molecular and genetic technological approaches can offer an important opportunity to supplement the current subjective symptom-based diagnosis [27]. However, the development of screening tests for psychiatric disorders is still remote. The only screening tests currently usable for psychiatric disorders are behavioral assessments, which are associated with an individual ’s higher risk in developing the disorder (e.g. family history of mental disorders; gender; substance abuse), and the investigation of discrete genetic alterations [27].

Familial or twin studies, indeed, have shown genetic predis- positions for PTSD or MDD [28], which highlight the role of genetic polymorphisms in some psychiatric disorders. In particu- lar, some polymorphisms of the dopaminergic and serotoniner- gic circuits have been found. A polymorphism for the dopamine D2 receptor seems to contribute to a PTSD predisposition [29], while, the serotonin transport gene is linked with more depres- sive symptoms and suicidal ideations [30]. The results of these early studies have not yet been replicated and their clinical implications remain premature. While these studies confirmed a possible implication of the dopaminergic and serotoninergic systems in psychiatric disorders, not even a single SNP has currently been associated with MDD or PTSD by large genomic- wide association studies. The lack of a direct link between single gene alterations and the disorders is probably due to the researcher ’s oversimplified models, which fail to take into account the interaction between brain, environment, and gene expression networks [31]. However, one of the principal neuronal deficits, which was firstly pointed as a primary deficit in depres- sion, was the serotoninergic neurotransmission, although this hypothesis was never found to reach a convincing scientific consensus [32]. A fundamental evidence for the original hypoth- esis of serotonin ’s role in depression derived from the mechan- ism of tricyclic antidepressant (TCA) on the reuptake of serotonin.

This gave rise to develop a number of antidepressants, such as the selective serotonin reuptake inhibitors (SSRIs), which, by blocking the serotonin transporter, enhance the activity of ser- otonin in depressed patients ’ brain and thereby improve their depressive symptoms [33,34]. However, SSRIs only improve symptoms in 50% of MDD and PTSD patients [35] and this disappointing result highlights that more and better biomarkers need to be discovered.

Given that PTSD and MDD are multifactorial disorders, it is important to establish a number of biomarkers and treatment targets for these disorders so that patients can be classified based on their specific biochemical deficits. Furthermore, this approach is relevant in that it may enable the stratification of patients ’ subpopulations, allows the design of better clinical trials and thus the testing of better treatments. Therefore, if a patient has a deficit in a specific neuroactive steroid level, an individually designed therapy will supplement that deficient

neuroactive steroid, its surrogate, or an agent that stimulates its synthesis. It will not provide a serotonergic molecule that may not help and may only result in unwanted side effects.

Recently, biomarker discovery for MDD and PTSD has sug- gested a number of neurochemical deficits in preclinical and clinical settings, including hypothalamic –pituitary–axis (HPA) alterations in response to stress, epigenetic changes, variations of immune response or signalling, and dysregulation of the endocannabinoid and neurosteroid systems, which will be examined in further detail in the sections to follow.

2. Biomarker candidates in PTSD and MDD 2.1. Epigenetic biomarkers

In PTSD and MDD, exposure to a trauma can epigenetically impact neuronal function and affect the neurophysiological and behavioral mechanisms of stress response and adaptation [36]. The phenotypes of PTSD and MDD emerge from the com- plex interaction between multiple genetic and environmental factors. Epigenetic changes consist of numerous biochemical processes, including DNA methylation and hydroxymethylation, histone posttranslational modifications (PTMs), and noncoding microRNAs. All these processes are influenced by the exposure to the environment and affect the transcriptional function of genes [37]. In absence of nucleotide DNA changes, epigenetic altera- tions can provide a molecular mechanism for the development of various phenotypes after traumatic events. However, trauma exposure does not necessary lead to the development of PTSD or MDD. Environmental factors that lead to psychiatric disorders can be precipitated by other factors, such as genetic predisposi- tion, timing of exposure or previous epigenetic modifications due to previous traumatic events [38,39]. To date, most epige- netic studies have examined the methylation status of cytosine residues in genomic DNA. DNA methylation, indeed, can be embedded by early life adversities or inherited through genera- tions [40 – 42]. Enzymatic methylation and demethylation pro- cesses dynamically regulate the methylation of target genes, although some stress-induced epigenetic changes may become permanent [43 – 45].

Exposure to early life traumas is very frequent in the psychiatric patients ’ history [ 46,47]. During childhood, the programming of the neurobiological system and the most relevant epigenetic alterations take place [48]. Thus, an early adverse experience, like child abuse and neglect, dramatically enhances an individual pre- disposition to develop depression or other psychiatric disorders later in life. Rodent studies that have been translated to humans showed that early life maltreatment alters the epigenetic program- ming of the HPA. For example, the histone PTMs affecting the epigenetic status of the glucocorticoid receptor (GR), gene NR3C1 in the hippocampus of the rat varies according to the level of parental care of pups [49]. This evidence has been confirmed by human studies in which suicide victims with childhood abuse history showed changes in methylation level of NR3C1 compared to suicide victims without reported childhood abuse [50].

Moreover, the amplitude of DNA methylation changes affecting

NR3C1 in adults with a psychiatric disorder diagnosis has been

correlated with the repetition and the severity of the maltreat-

ments during childhood [51].

Beside the HPA axis, the epigenetic status of other systems involved in neuropsychiatric disorders has been investigated in relation to early life stress, such as the brain-derived neurotrophic factor (BDNF) gene. The level of methylation of the BDNF gene is higher in peripheral blood cells of individuals who received low parental care [52]. Moreover, a hypermethylation of this gene has been detected in blood of depressed patients [53].

The advancement in genome-wide analysis has led the research beyond the gene-candidate approach toward a more comprehensive mapping of the neuroepigenome. The sensitivity and stability of the epigenetic alterations make them promising biomarker candidates. For example, a longitudinal study showed an association between psychiatric disorders, the amygdala – hippocampus volume ratio and the methylation of the SP6 gene, making the DNA methylation an epigenetic predictor of a specific disorder [54].

Currently, besides the status of the DNA methylation, the investigation of microRNAs (miRNAs) is becoming an increas- ingly focus of investigation for their functional relevance in several pathological conditions. miRNA are members of a class of noncoding RNA, transcribed from introns or exons of noncod- ing RNA by polymerase II [55]. Preclinical and clinical studies have shown an association between miRNA –mRNA interactions and depression or PTSD. A polymorphism of miR-30e, indeed, is positively correlated with the symptomatic onset of depression [56,57]. In the blood of male combat veterans with PTSD, eight miRNAs have been found to be differentially expressed, with four upregulated and four downregulated, compared to other healthy military personnel [58]. In the peripheral blood mono- nuclear cells, Bam and colleagues found higher DNA methylation in male combat veterans with PTSD and identified an altered expression of 190 miRNAs, among which 183 were downregu- lated [59]. Male and female combat veterans with PTSD showed

decreased miR-125a and miR-181c levels [60], while mi-R3130-5p is decreased in patients with PTSD and comorbid depression of both sexes [61]. Both the increase and the decrease of miRNA function are associated with a depressive psychopathology. The overexpression of miRNA leads to an excessive silencing of sev- eral targets, while impairment of miRNA expression leads to over-translation of mRNA, affecting neuronal physiology [55].

The capability to mediate multiple targets makes miRNA a potential therapeutic strategy to recover from the chemical imbalance of patients with psychiatric disorders, but caution is necessary to avoid unexpected effects of altered miRNA expression.

Most of the epigenetic studies have failed to adequately address the timing of exposure to trauma, the temporal rela- tionship with the epigenetic alterations and the onset of PTSD or MDD. Genome-wide studies highlighted the importance of early trauma in the epigenetic changes: patients with different traumatic histories showed distinct DNA methylation profiles and different blood gene expression. Understanding the genetic vulnerability of factors that contribute to psychiatric illness may enable the identification of individuals who fail to cope with traumatic events (depicted in Figure 2). Traumas clearly induce epigenetic changes with short- and long-term effects on neuronal function, brain plasticity, and behavioral modifications [36,62,63]. The reversibility of the epigenetic alterations makes them potential biomarker candidates for the development of new treatments for psychiatric disorders, as demonstrated by studies in rodents [64].

2.2. Chronic stress and role of the HPA axis

Aside from a genetic vulnerability, one of the major causes of depression and PTSD is the exposure to chronic and repeated

Figure 2. Genomic and epigenetic role in the development of psychiatric disorders.

The early life experiences, particularly early life adversities, may lead to epigenetic alterations. In absence of traumas and stressful conditions, there is an increased possibility of developing

resilience in adult life. The exposure to early life adversities in childhood may result in a phenotype that is more susceptible to psychiatric disorders following a more recent exposure to

a stressor. Nevertheless, even trauma-exposed adults who have not experienced adversities during early life are susceptible to develop PTSD and/or MDD.

stress. Prolonged stress leads to alterations of behavior and physiology of both humans and rodent models of depression and PTSD [65,66]. The stress response is mediated by the HPA axis; whose role in MDD and PTSD has been consistently demonstrated [67]. The stress response is mediated by the rapid activation of the sympathetic nervous system, which leads to the release and increase of noradrenaline and adre- naline. Blood concentrations of adrenal glucocorticoids also rise after exposure to stressors. Alterations of the corticotro- pin-releasing hormone (CRH) function have been reported and enhanced levels of CRH have been found in the cere- brospinal fluid (CSF) of depressed patients [68]. The increased release of CRH results in a greater secretion of adrenocorti- cotropic hormone (ACTH) and in increased glucocorticoid synthesis, in particular cortisol, which has an inhibitory feed- back on CRH and ACTH through the GR and the mineralo- corticoid receptor (MR). The affinity of these two receptors for glucocorticoids is different: MR maintains its activation for 1 h after the hormone release, while GR is progressively activated during stress. When the stress is prolonged, gene- mediated corticosteroid effects take over through the tran- scriptional regulation of specific sets of genes by the MRs and GRs. The functions of different proteins, indeed, are altered by the corticosteroid-mediated stress response. Of note, patients with MDD and PTSD show enhanced levels of cortisol in saliva, plasma and urine, and an increased size and activity of pituitary and adrenal glands [69]. The basal cortisol concentration has been associated with the induction of hippocampus long-term potentiation (LTP) that is implicated in memory formation [70]. On the contrary, stress, new envir- onment, and high corticosteroid levels impair LTP and facil- itate long-term depression [71]. The inability to face stressful life events leads to hypersecretion of corticosteroids, increas- ing the risk for psychiatric disorders in vulnerable individuals [72]. Chronic stress exposure induces an enhanced produc- tion of CRH mRNA in the paraventricular nucleus (PVN). The exact mechanism driving the increases in CRH gene expres- sion is not completely understood, probably, it involves the repeated activation of cAMP [73]. In healthy individuals under chronic stress conditions, CRH coordinates adaption by inducing release of corticosteroids; however, this mechan- ism fails during depression due to an altered expression of GR levels and to a disruption of the physiological activity of the HPA axis [74]. The receptor subtype for CRH, CRHR1, seems to have important implications on anxiety and depres- sion [75]. Papiol and colleagues showed that homozygous patients for TT in the SNP of CRHR1 gene, rs 110402, present an earlier onset of the disease and thus this SNP has been suggested as a biomarker of acquired vulnerability [76].

Several studies evaluated the pituitary negative feedback inhibition of the HPA axis by stimulating the GR by the admin- istration of the synthetic glucocorticoid, dexamethasone (dex).

The dexsuppression test was elaborated as a diagnostic test for depressed patients with comorbid melancholia, but with- out consistent results because the same neuroendocrine alterations in this subgroup also occur in patients with differ- ent diagnosis [77]. Patients with different mood disorders show elevated plasma cortisol concentrations after an oral

administration of dex, presumably due to impaired functions of GR and MR in the pituitary [67]. By contrast, in healthy individuals, dex leads to an inhibition of the HPA axis and to decreased cortisol levels for up to 24 h [78]. The high cortisol non-suppression after dex administration has been linked with early relapse and poor clinical response in MDD [79].

Furthermore, a refined test combining dex administration and CRH stimulation (the dex/CRH test) showed that both CRH and vasopressin drive HPA activity in depressed patients [80]. Elevated cortisol response to the dex/CRH test has been observed in MDD patients. Antidepressant treatments reduce the release of cortisol after the dex/CRH test, probably because antidepressants improve corticosteroid receptor func- tion and can regulate HPA axis activity [67]. All these findings led to the hypothesis that a restoration of corticosteroid receptor function results in a better HPA axis regulation, which precedes a clinical amelioration. In healthy individuals with high genetic load for depression, the dex/CRH test showed higher cortisol response than in individual without this genetic background [81]. This evidence highlights that changes in the HPA axis are genetic traits that increase the risk for MDD [74]. The dex/CRH test failed to show abnormal- ities in HPA axis function in male veterans with PTSD com- pared to trauma-exposed male veterans without PTSD [82]. In PTSD patients with comorbid MDD, the ACTH response to the test is attenuated compared to PTSD patients without MDD, suggesting the presence of subpopulation in PTSD with dif- ferent HPA axis regulation. By comparing studies on individual with PTSD or with MDD, the dex administration leads PTSD patients to a higher cortisol suppression, while in MDD patients leads more frequently to cortisol non-suppression [82]. Altered HPA axis function in PTSD may be more related to reduced basal cortisol level and enhanced CRH activity, while in MDD, HPA axis abnormalities are probably due to reduced GR responsiveness and increased cortisol levels [74,83].

The glucocorticoid system is a potential relevant target for MDD and PTSD therapeutics. For example, GR sensitiv- ity is regulated by FKA-binding protein 51 (FKBP5) and depressed patients exhibit decreased GR sensitivity with a substantial reduction of FKBP5 mRNA expression. The FKBP5 gene transcription is dependent on GR activation with a feedback modulation that regulates GR sensitivity.

After an administration of dex, depressed patients showed

a GR-mediated alteration in gene expression compared

with healthy controls [84]. However, the FKBP5 gene

appears to play a primary role in psychiatric pathologies

and it is an important predictive biomarker. For example,

FKBP5 polymorphisms associated with early traumatic

events, like childhood abuse, predict adult major depres-

sion, and PTSD [85]. Of note, adversities during childhood

influence the transcriptional activity and the state of HPA

axis genes implicated in the response to stress. Moreover,

the onset of PTSD after trauma exposure is associated with

pre-traumatic biomarkers, such as higher GR number in

peripheral blood monocytes or low mRNA levels of the GR-

inhibitor FK506-binding protein 5 (FKBP51), which reveal

the level of sensitivity to stress [86 – 88].

A study of Binder and colleagues showed that three poly- morphisms of this gene are potential diagnostic biomarkers because significantly associated with an enhanced FKBP5 expression, which regulates the HPA axis function. Patients with these polymorphisms show less hyperactivity of the axis during depression. Moreover, rs1360780 was strongly asso- ciated with faster antidepressant response in patients, which plays a pivotal role as biomarkers to predict the treatment outcome [89].

The interaction between early traumas and polymorphisms determines the methylation state of the gene and regulates the sensitivity of FKBP5 to GR regulation [39]. Remarkably, the severity of PTSD is associated with the level of FKBP5 gene expression; low expression of this gene is linked to low plasma cortisol [90]. Several studies suggest that the low cortisol levels can regulate the FKBP5 expression through changes in the GR responsiveness [91]. By contrast, a study on PTSD patients exposed to the World Trade Center attack showed that alterations in FKBP5 may facilitate GR responsiveness, deceasing cortisol levels [90]. However, the results of this works are limited by the small subsample of patients (20 Caucasians) with current PTSD, which make it hard to distin- guish between genes associated with risk, recovery, and resi- lience. For this reason, the findings of this early study should be replicated including a cohort of remitted PTSD patients and focusing on the function of other relevant genes, such as GR gene or the nuclear factor I/A, a transcription factor that acts in concert with GR, whose expression has been found altered in PTSD patients [90].

In another recent study, Yehuda and colleagues suggest two possible stable epigenetic biomarkers for PTSD: GR and the FKBP5 gene methylation. PTSD patients, classified as responders, showed a greater average number of methylated site at pretreat- ment assessment compared to nonresponders, but the methyla- tion of the GR gene was not altered at posttreatment or follow- up. On the contrary, the methylation status of FKBP5 gene decreased in association with recovery. The GR exon 1F promoter methylation predicted the outcome of the treatment, while, the cytosine methylation of FKBP5 promoter seemed to be asso- ciated with treatment outcome [92]. It is important to highlight that the GR gene methylation does not change over time sig- nifying that early environmental experiences cause a durable epigenetic modification of this gene [93]. In animal studies, it has been observed that variations in maternal care lead to a different methylation pattern of the GR gene with lasting effects on GR responsiveness in adults [94]. In humans, child abuse is linked to enhanced methylation of GR exon 1F promoter in leukocytes and in the postmortem hippocampus [50].

The early life experiences have a strong impact on gene expression and early life adversity can influence both GR and FKBP5 methylation. In PTSD, the GR promoter methylation induces an enhanced GR sensitivity with low levels of gluco- corticoids, which decrease FKBP5 gene expression. This dimin- ished FKBP5 gene expression could sustain the increased sensitivity of GR. Thus, the methylation of the FKBP5 promoter leads to an increase of this gene expression and, conse- quently, to a reduced GR sensitivity. Coincidently, patients who respond to treatment show a decrease methylation of the FKBP5 promoter and of GR sensitivity.

Methylation or demethylation of a number of other genes and their expression patterns are influenced by glucocorti- coids. This includes genes linked to the synthesis of BDNF, which affects neurogenesis, of neuropeptides, such as neuro- peptide Y(NPY) and α-MSH, or of enzymes involved in bio- synthesis of neurohormones, such as the GABAergic neuroactive steroids [95 – 97].

2.3. The immune system modulation of stress responses An inflammatory response characterized by increased number of granulocytes and monocytes, enhanced acute phase reac- tants and inflammatory cytokines levels has been demon- strated in individual with MDD compared to healthy controls [98 – 100]. Furthermore, the role of cytokines (interleukin IL-6, IL-1, and TNF- α) in the regulation of emotional behavior and memory formation during stressful life events, and their inter- action with neurogenesis, glutamate and GABA signaling or changes in LTP has been documented [101,102].

The most promising inflammatory biomarkers in serum of sub- jects with psychiatric disorders are pentraxin C-reactive protein (CRP), tumor necrosis factor alpha (TNF- α), and IL-1 and IL-6 [ 103].

CRP, an acute-phase protein, produced by the liver, is found in plasma in response to inflammation and could be an important biomarker for psychiatric disorders, in addition to participating in inflammatory processes [104]. Indeed, an increase in CRP level was shown in patients with PTSD or in suicidal patients [105,106]. Some recent studies showed that patients with CRP levels above 10 mg/l responded better to treatment with TCAs or SSRIs than to psy- chotherapy alone [107] and that patients with low CRP levels (<1 mg/l) showed more consistent treatment response to escita- lopram than to nortriptyline [108]. Taken together, these findings suggest that CRP may be an intriguing predictor of treatment outcomes that could lead to developing a personalized treatment approach. In depressed patients, an increased level of plasma CRP and IL-6 [109], and the serum or plasma levels of IL-1 β and IL-6 are often related to antidepressant treatment, given that their levels inversely correlated with treatment response [110].

The investigation of mRNA levels of cytokines in MDD patients showed an increase of several inflammatory factors, such as IL-1 β, IL-6, and TNF-α. Furthermore, two SNPs of IL-1β gene, rs16944 and rs1143643, were strongly associated with decreased responsiveness to antidepressants and the fMRI- analysis showed reduced amygdala hyperactivity to emotional stimuli [111]. A SNP was also discovered for the IL-6 gene, rs1800795, a potential diagnostic biomarker, which is asso- ciated with enhanced levels of plasma IL-6. Furthermore, its interaction with additional stressors increases the risk of MDD development or of other psychiatric disorders, such as schizo- phrenia [112,113]. A study by Sukoff Rizzo and colleagues, in rodents, demonstrated that an increased IL-6 level in the brain results in development of depressive-like behavior and the activation of IL-6 inhibits the antidepressant effect of SSRIs [114]. This finding may in part explain why some antidepres- sants fail to induce beneficial pharmacological effects in patients with MDD and PTSD.

Another cytokine that is increased in the plasma of

depressed patients is TNF- α. Results show that failure to

normalize its levels also correlated with failed response to SSRIs [98,115]. In particular, recent studies suggest that responders to antidepressants show a lower expression of TNF- α. Lower expression of this cytokine (30%) was observed after successful treatment with escitalopram [116]. A meta- analysis study showed a specific profile in the changes of peripheral cytokines levels in MDD patients, which showed elevated concentration of IL-6, TNF- α, IL-10, sIL-2R, CCL-2, IL- 13, IL-18, IL-12, and TNFR-2, while interferon- γ (IFN-γ) was slightly reduced [117]. A study by Goldsmith and colleagues [118] investigated the blood cytokine alterations in three dif- ferent psychiatric disorders: schizophrenia, bipolar disorder, and depression. They found strong similarities among the changes in cytokine levels, raising the hypothesis of a common pathway for immune dysfunctions in these disor- ders. Interestingly, they also investigated the effects of anti- depressant treatments on blood cytokines levels, showing also similarities in the changes of peripheral immune molecule levels. For example, in patients with MDD and schizophrenia, IL-6 decreases, while TNF- α fails to change.

Inflammatory responses play a pivotal role in PTSD as demonstrated by cytokinemia and meta-analysis studies revealed that PTSD patients have increased levels of TNF- α, IL- β and IL-6 in plasma [ 119]. Furthermore, PTSD risk and PTSD cases are associated with an increase of the expression of genes linked to innate immune responses mediated by inter- feron signaling. This evidence shows the importance of differ- ences in innate immune factors for the development of PTSD [120]. A study by Pervanidou et al., showed an increased serum IL-6 concentration, measured 24 h after motor vehicle accidents, in children and adolescents who developed PTSD [121]. A longitudinal follow-up study revealed a normalization of IL-6 levels over time, validating the predictor role of IL-6 for PTSD. Moreover, other studies confirmed an increased IL-1 β and IL-6 plasma levels and further showed that IL-1 β is posi- tively correlated to PTSD symptom duration, while IL-6 corre- lated to the PTSD symptom severity [119]. Contrarily, the proinflammatory cytokine IL-18 has been found downregu- lated in PTSD patients by a cDNA microarray investigation.

The increased IL18 gene methylation explains the lower expression of this cytokine, which plays a neuroprotective role by inducing IFN- γ activity [ 122].

Whereas most PTSD studies have focused on the innate immune system, the acquired immune system can also be a mediator of risk and resilience of PTSD [123,124]. The regulatory T cells, such as CD4+ T, support CNS function, interacting with brain response to stress and promoting neuroprotection and neu- rodegeneration [125]. In chronic PTSD, there is a profound altera- tion of the composition of the peripheral T cell compartment [126].

Changes in T cell phenotype and increased differentiation of T cells are associated with PTSD and an elevated differentiation rate is taken as an early aging indicator of the immune system [127].

Combat veterans with PTSD showed a preponderance of CD4+ T helper 1 over the CD4+ T helper 2 cells, which is strongly correlated to enhanced IFN- γ plasma levels [ 128].

These findings collectively suggest that proinflammatory mar- kers may be useful to predict treatment outcomes and for diag- nosis of the pathology. Whether the enhancement of proinflammatory markers reflects a neural dysfunction or whether

these molecules play a role in the pathogenesis of psychiatric disorders is unclear, however, their alteration may be valuable as a marker to determine the most appropriate treatment.

2.4. Neuroplasticity

PTSD and MDD are associated with low levels of neurotrophins, including BDNF, which play a fundamental role in neuronal growth, synaptic maturation and plasticity [129]. BDNF is funda- mental for the development of the nervous system and decreased serum levels of this neurotrophin and/or of its recep- tor expression, TrkB, may be a useful predictor for dysfunctional behavior and in particular for suicidal ideations [105]. Studies in rodent models show a decreased BDNF mRNA expression in the hippocampus during chronic and acute stress, with consequent effects on hippocampal neurogenesis [130]. Furthermore, the reduction of BDNF mRNA expression has been associated with increased IL-6 expression [131]. Importantly, both glucocorti- coids and proinflammatory cytokines have been often linked to a reduction of BDNF levels and to a diminished neurogenesis resulting in dendritic atrophy [132,133]. The mechanism by which proinflammatory cytokines could affect BDNF expression is still unclear. However, animal studies showed that IL-1 β downregulates BDNF expression in rat hippocampus, probably by an indirect mechanism that relates to regulation of gluco- corticoids [134]. Overall, depressed patients show a reduction of BDNF mRNA expression, with increased levels that correlated with symptom improvement in antidepressant responders [135]. A recent meta-analysis investigation has suggested that BDNF is a promising biomarker for MDD patients relevant to predict clinical improvement [136].

In contrast to the observations in MDD patients, the BDNF levels in the serum of subjects with PTSD are controversial.

Some researchers reported that it increases in PTSD and it is

even higher after traumatic events [137], while others showed

lower levels [138]. Berger and colleagues found that serum

BDNF level is a good predictor of the treatment outcome in

PTSD: lower serum BDNF is a reliable marker for a successful

treatment response to escitalopram in patients. The apparent

discrepancy in BDNF results in PTSD patients may be

explained by findings that the inhibition of BDNF activity

could have mood-regulatory activity in some animal models,

which suggests a biphasic concentration-dependent activity of

BDNF [139]. The role of BDNF in the mesolimbic area is in

contrast to the role of BDNF in the hippocampus, because

high levels of BDNF in the dopamine pathway may be pivotal

for the onset and maintenance of PTSD. BDNF, indeed, acts as

a strong excitatory neurotransmitter, which evokes a rapid

postsynaptic depolarization, promoting the dopamine release

in the nucleus accumbens (NAc) [140]. In mice, the role of

BDNF in this pathway is to maintain social avoidance behavior

induced by the defeat stress paradigm. Hence, peripheral

BDNF levels can provide an indicator of the activity of the

dopaminergic system in the NAc and could be a predictor of

poor response to treatment [139]. Furthermore, this discre-

pancy may also result from the methods used or differences

in variables, including age or the subpopulation considered, all

aspects that can alter the expression of BDNF and make the

interpretation of results difficult as well as limit the applicabil- ity of this potential biomarker.

Polymorphisms of the BDNF gene have also been investi- gated, the rs6265, resulting from a substitution of Val66Met, is frequently linked to a higher risk in developing affective disor- ders [141,142]. This SNP influences hippocampal volume and memory and may increase the susceptibility to both depression and PTSD [143]. Furthermore, the frequency of Val66Met is two times higher in PTSD patients than in healthy controls [144].

Stress and the Val66Met-allele seem to interact; a study on healthy European volunteers suggests that this interaction may result in development of depression and anxiety [145].

Whereas the association of BDNF levels and the symptoms of MDD and PTSD is still controversial, its role in the response to antidepressant treatment is clearer [146]. Several studies have demonstrated that chronic treatment with different SSRIs increases BDNF mRNA and protein expression in hippocampus and cerebral cortex [147,148]. Furthermore, all pharmacological classes of clinically used antidepressants increase TrkB autopho- sphorylation and signaling in hippocampus and forebrain [149,150]. Similarly, acute treatment with ketamine induces an increase in BDNF mRNA and TrkB phosphorylation [151]. Rodent studies show that BDNF and TrkB signaling is necessary for the behavioral effects of antidepressant drugs [146]. Indeed, reduc- tion of BDNF levels in forebrain regions and inhibition of TrkB signaling in the dentate gyrus block the behavioral effects of antidepressants [150]. This evidence led to the hypothesis that antidepressants by stimulating neurotrophins promote neuronal plasticity and improve behavioral impairment in patients with MDD or PTSD [152]. In PTSD, the correlation between antide- pressant effects and BDNF is more questionable, because few studies consider these three factors together. Berger et al.

showed that despite an improvement of PTSD symptoms due to escitalopram, BDNF levels in blood failed to change [139]. In a case report of Hauck et al., PTSD patients, who were treated with sertraline for 6 weeks, showed a reduction of symptoms, but no increase in serum BDNF levels [153].

Measuring the levels of BDNF in blood may advance the understanding of the neurotrophins ’ role in mood disorders, whether it may offer a potential biomarker to monitor treat- ment response in PTSD patients remains unclear.

2.5. The NPY implications in resilience

NPY is an important mediator in the regulation of the stress responses [154]. NPY and its receptors play a fundamental role in decreasing fear, anxiety, and also improving memory pro- cesses [155,156]. Stress modulates NPY expression in the brain, but the duration and type of stress influence the magnitude and direction of NPY expression change [157]. Rodent studies showed that foot-shocks increase NPY gene expression while acute restraint stress exerts the opposite effect in the amyg- dala [158,159]. Moreover, acute but not chronic stress enhances NPY transcription levels in the hypothalamus, sug- gesting a possible habituation to repeated stress exposure [157]. NPY participates in the regulation of the HPA axis by interacting with the PVN of the hypothalamus and with the action of CRH [160]. Stimulation of NPY Y1 receptors in the hippocampus of rodents appears to inhibit the HPA axis,

although some studies have reported different results [161 – 163]. For instance, the administration of the Y1 receptor antagonist, BIBP 3226 in the amygdala exerts an anxiogenic effect, while intracerebroventricular injections of a specific Y1 agonist ([D-His(26)]NPY) have an anxiolytic effect [126,164].

Another mechanism of action of NPY-induced behavioral changes emerged with the Y1 agonist (Leu ³¹ ,Pro ³⁴ ), which reduced the amplitude of NMDA-evoked excitatory postsynap- tic currents and increases the inhibitory current magnitude induced by GABA _A receptor activation [165].

The involvement of NPY in stress response and by improv- ing emotional behavior is further analyzed in rodent stress models. Rats exposed to a predator scent stress protocol, an animal model of PTSD, showed that the animals with the lower NPY levels had the more significant behavioral disrup- tion, and treatment with NPY reduced the altered behaviors [166]. The use of Y1 receptor antagonist, BIB03304 led to a behavioral disruption confirming the pivotal role of Y1 receptor in behavioral dysfunction [166]. Other animal studies have showed that NPY supplementation after trauma expo- sure prevents the development of PTSD-like behaviors. For example, the intranasal administration of NPY by increasing the concentration of NPY in the CNS, devoid of side effects, led to behavioral improvements in animal models for PTSD, such as the single prolonged stress and the predator exposure stress [167,168].

Evidence demonstrates that NPY is relevant in PTSD and MDD pathophysiology. Plasma NPY levels in humans increase after the exposure to acute stress. The enhancement of NPY concentration is positively correlated with the stress-induced increase of cortisol salivary levels [169]. During stress, the increased release of NPY may provide a positive psychological response, preventing the anxiogenic effect induced by CRH. Moreover, the peripheral increase of NPY detected in plasma may reach the brain and exert central effects. Interestingly, individuals who experienced dissociative PTSD symptoms, 1 week before stress exposure, showed a lower release of NPY in response to stress [169]. This evidence suggests that NPY may be a relevant biomarker to pre- dict the ability of an individual to cope with stressful events.

Veterans with PTSD showing symptoms of dissociation exhibit

a reduced capacity to release NPY [170]. Thus, the altered release

of NPY can influence the risk for the development of PTSD. Other

studies showed that NPY concentrations in the CSF and plasma are

reduced in patients with PTSD [170 – 172]. In particular, a study by

Rasmusson et al. [170] found that basal and yohimbine-stimulated

NPY plasma levels are lowered in PTSD subjects. Reduced NPY

levels in plasma were found also in combat-exposed individual

with PTSD [173]. However, NPY levels were also reduced in com-

bat-exposed individuals without PTSD, showing that the trauma

itself could be a factor, in this study, in the regulation of NPY

concentrations [173]. However, later studies observed opposite

results, showing both trauma-exposed PTSD patients with no

alterations of NPY levels [174] and combat-exposed veterans with-

out PTSD with a higher NPY expression [175]. Because of the

questionable reliability of plasma NPY concentrations, other stu-

dies started to focus on its CSF levels. Sah and colleagues showed

that combat-exposed veterans with PTSD have lower NPY levels in

the CSF in comparison to trauma-exposed veterans without

PTSD [172].

The abnormalities in NPY concentrations are of interest because NPY seems to be implicated in resilience development and prevents trauma-induced PTSD. Genetic evidence supports this role of NPY: Individuals with lower-haplotype NPY expression show exaggerated amygdala reactivity, which reflects an over- reaction to stress [176]. However, direct associations between NPY gene polymorphisms with PTSD have not been observed.

Several SNPs of the NPY gene have been studied and, for its role in stress responsiveness, rs16147 is a promising biomarker for vulnerability to anxiety and depressive symptoms [177]. The NPY SNP rs16147 is highly related to the variation of the NPY levels and is associated with a reduction of NPY expression [176].

Studies on young adults showed an interaction between rs16147 and early life adversity. In addition to rs16147, the poly- morphisms rs3037354 and -1002T>G are important for the stress response. The SNP rs3037354 is associated with higher plasma NPY and changes in the expression of GR signaling [178]. On the contrary, loci in the NPY promoter (1002 T>G) induce a lower NPY expression detected in CSF, which is associated with hyperarou- sal during the exposure to stress [179].

Measurements of NPY in CSF of depressed patients led to overall divergent results. Several studies reported significant CSF NPY reduction in individuals with MDD, while other studies failed to find differences or showed higher NPY levels in CSF of depressed individuals [180]. The individual ability to adapt to stress and/or the genetic history may explain the differences found in NPY levels of MDD patients. Studies on NPY in indivi- dual with PTSD and comorbid MDD are under-investigated.

Only a study in PTSD patients with comorbid depression by Sah et al. showed an inverse correlation between NPY concen- tration in CSF and PTSD diagnostic CAPS scores, but no correla- tions were found with Beck Depression Inventory (BDI) [172].

Altogether, whether NPY could be a valuable therapeutic target for PTSD and MDD and whether it may help facilitate resilience in vulnerable subjects remains a matter of debate [180,181].

2.6. The role of endocannabinoids and allopregnanolone in MDD and PTSD

2.6.1. The biosynthesis of neuroactive steroids

Neurosteroids and neuroactive steroids modulate neuronal functions after binding to nuclear intracellular receptors and by acting as transcription factors with important implications in modulation of gene expression [182]. Neuroactive steroids can also act via nongenomic mechanisms as potent modula- tors of ligand-gated ion channels, including GABA _A and NMDA receptors [183,184]. Neuroactive steroids play a role in stress response, are implicated in neuropathology of an array of psychiatric disorders and may mediate the response to several psychotropic treatments [185].

Neurosteroids, allopregnanolone (Allo) and its stereoisomer, pregnanolone (PA), are synthesized in glutamatergic corticolim- bic neurons, including cortical and hippocampal pyramidal neurons, and pyramidal-like neurons of the basolateral amyg- dala [186,187]. They rapidly modulate neuronal excitability by acting as potent positive allosteric modulators of the action of GABA at GABA _A receptors and they are responsible for the fine- tuning of the receptor for GABAmimetic, agonists and positive

allosteric modulators [188 – 191]. Recent finding in the field have suggested that the sulfated congeners of these neurosteroids, e.g. PA sulfate, act as inhibitors of tonic rather than phasic NMDA-mediated neurotransmission [192].

Downregulation of neurosteroid biosynthesis, which includes Allo and PA levels and their biosynthetic enzyme 5 α- reductase type I and 3 α-hydroxisteroid dehydrogenase (3α- HSD), is strongly associated with major depression and PTSD (for a review please see [193,194]). Specifically, patients with depression show serum, plasma, CSF, and brain reductions of Allo levels and/or biosynthesis, which may become important diagnostic biomarkers [195 – 198]. Likewise, depression and anxiety symptoms in both anorexic and obese females or during pregnancy and postpartum are associated with down- regulated Allo levels [199,200]. The levels of Allo in the CSF are 40 –60% decreased in patients with unipolar major depression and premenopausal women with PTSD [197].

The lowest levels were found in the PTSD patients with comorbid depression [201]. Altered Allo levels have been observed both in serum and CSF in several other neuro- pathologies, including postpartum depression and drug addiction [197,202]. Women with PTSD show lower Allo con- centration in the CSF and serum, while progesterone and the immediate Allo precursor, 5 α-DHP fail to change, pointing to a possible deficit in the enzyme 3 α-HSD [ 203]. Likewise, in PTSD males, the CSF Allo levels decrease, probably because of deficits of 5 α-reductase type 1, and are negatively corre- lated with PTSD symptoms [204]. Moreover, a SNP in the 5 α- reductase type II gene is linked to enhanced risk for PTSD in men [205]. 5 α-reductase type II is preferentially expressed in the periphery in the adrenal cortex [206]; however, peripheral GABAergic neuroactive steroids, including THDOC, are chan- ged during stress and may access and influence corticolimbic circuitry [207]. Thus, the concentration and, in particular, the ratio of Allo with other neuroactive steroid levels and deficits in the enzymatic pathway may unveil sex-related biomarkers for PTSD and MDD, as well as provide a target for novel therapeutics to improve MDD and PTSD patients. Studies in depressed patients with low Allo concentrations in CSF and plasma showed that after SSRI treatment, increased Allo level correlated with improvement of depressive symptoms [196,197].

The plasma levels of the GABA _A antagonist and neuroactive steroids, dehydroepiandrosterone (DHEA) and its sulfate deri- vatives, DHEA sulfate (DHEAS) are currently being investigated as potential biomarkers for anxiety, MDD, and PTSD. DHEA facilitated excitatory NMDA receptor function and plays a role in the inactivation of cortisol in its metabolite cortisone [208]. The DHEAS concentration and the ratio of DHEAS to cortisol predicts the severity of symptoms of PTSD and depres- sion in patients [209]. In the CSF of women with PTSD, correla- tion between the ratio of DHEA to Allo levels and their symptoms was observed [201]. This suggests a role in the balance between excitatory and inhibitory neurotransmission and the severity of the pathology.

In mouse models of depression, induced by protracted social

isolation stress, a ownregulation of Allo levels in corticolimbic

neurons was observed and resulted from a decreased expres-

sion of 5 α-reductase type I [ 210]. Furthermore, socially isolated

(SI) mice show increased aggression, anxiety-like behavior and exaggerated contextual fear responses [211,212]. In another model of PTSD, the single prolonged stress (SPS) mouse, down- regulated cortical Allo levels were associated with enhanced anxiety-like behavior and enhanced contextual fear responses.

Importantly, similarly as in PTSD patients [213], stress induces changes in GABA _A receptor subunit composition in SI mice.

Specifically, it increases expression of α4 and δ subunit that are: (1) mainly expressed in the extrasynaptic GABA A receptor;

(2) show an increased sensitivity for neurosteroids; and, impor- tantly, (3) fail to bind benzodiazepines, and therefore result in inefficacy to respond to their pharmacological action [202].

These findings are strikingly consistent with dysfunctions observed in PTSD patients. PET studies show PTSD patients have decreased benzodiazepine binding sites and lack of response to benzodiazepines [213]. Collectively, PTSD, like

MDD, shows a downregulation of Allo biosynthesis; however, this and its interface with changes in GABA A receptor subunit expression and lack of response to benzodiazepines points to a biomarker axis (Figure 3), which may be specific for PTSD (discussed in [193]).

In SI mice administration of Allo or its analogs (ganaxolone, BR351, and BR297) reduces behavioral dysfunction [193,214].

Furthermore, SSRIs given at low non-serotonergic doses act as selective brain steroidogenic stimulants (SBSSs), upregulate Allo levels, and improve fear responses, anxiety-like behavior and aggression in Allo-deficient SI mice [215]. Consistently, in recent clinical trials, intravenous Allo (i.e. brexanolone; SAGE 547) or an orally active Allo ’s analog (SAGE 217) showed increased efficacy against symptoms of postpartum depression (PPD) and MDD compared to that of placebo [216]. PPD patients who received intravenous infusions of Allo showed a rapid and long lasting

Figure 3. Potential role of endocannabinoids and neurosteroids as peripheral biomarkers for PTSD and MDD.

The schematic representation shows the mechanisms for the neurosteroidogenic action of the endocannabinoid-like, ethanolamides (e.g. PEA) in the brain (left panel). PEA and OEA

activate the intracellular peroxisome proliferator-activated receptor (PPAR)-α, which heterodimerize with the retinoid X receptor (RXR). The PPAR-α and RXR complex binds to the consensus

regions of target gene promoters and initiates transcription. PEA, through the activation of PPAR-α, can enhance the expression of corticolimbic allopregnanolone (Allo) biosynthetic

enzymes, including CYP11A1 and 5α-reductase type I, resulting in an enhanced neurosteroid biosynthesis (e.g. Allo) [278]. Rodent studies confirmed that the administration of PEA or of

PPAR-α agonists improve emotional behavior by increasing Allo levels [193]. PEA levels and probably expression of PPAR-α are influenced by stress, which may negatively affect Allo’s

biosynthetic enzyme expression and Allo levels. Allo and its stereoisomer pregnanolone (PA) are primarily synthesized in glutamatergic neurons and play a central neuromodulatory role in

facilitating the action of GABA at GABA

receptors (a primary target of anxiolytics), and a role in the fine-tuning of the receptor for agonists and GABAmimetic agents. The finding that Allo

facilitates the efficacy of GABA

receptor allosteric modulators substantiates its endogenous neurophysiological relevance. Allo and PA binding at GABA

receptors result in the

improvement of behavioral responses, including anti-aggressive, anxiolytic, and anti-fear actions. Sulfated neurosteroids may act on NMDA receptors (bottom, left pannel). The binding

of sulfated Allo and PA (PA-S) inhibits tonic-activated NMDA receptor neurotransmission, which results in important downstream effects on neuroplasticity, memory formation, and learning

processes with a high relevance for neuroprotection and cognitive processes [192,193]. The altered levels of endocannabinoids, endocannabinoid congeners, and neurosteroids in the blood

and/or CSF of PTSD and MDD patients are represented in the right panel. It is evident that the abnormal concentrations of endocannabinoids and neurosteroids in PTSD and MDD show

common alterations. The discovery of specific dysfunctions in the peripheral tissue of individuals can provide biomarkers to diagnose and treat PTSD and MDD. Moreover, the relation of

several biomarkers, by assessment of a biomarker axis, may help identify subpopulation of patients within one psychiatric disorder and may enable selection of individual-based therapeutic

strategies.

remission of depressive symptoms in 70% of treated versus only 9% of placebo-treated patients.

Likewise, the 18kDa translocator protein (TSPO), which gates the entry of cholesterol from the cytosol into the inner mitochon- drial membrane to initiate neurosteroidogenesis, resulted in a useful PTSD therapeutic target [217]. Drugs that act at TSPO stimulate downstream Allo levels in the brain of SPS mice and rescue behavioral dysfunctions [218]. By this mechanism, TSPO ligands promote anxiolytic effects in humans [218]. For example, the ligand, XBD173 potentiates GABA-mediated neurotransmis- sion through the induction of neurosteroidogenesis [219].

XBD173 counteracts panic attacks in rodents and exerts anti- panic activity in humans [219]. Hence, TSPO drugs are fast- acting anxiolytics, devoid of tolerance liabilities and with fewer benzodiazepine-like side effects, such as tolerance, dependence, and withdrawal symptoms. Moreover, Ströhle and colleagues [220] showed an altered benzodiazepine-GABA _A receptor func- tion in patients with panic disorder, which suggests neuroster- oid-based therapeutics that act on GABA _A receptors may offer a treatment advantage over benzodiazepines for these patients [219]. In preclinical studies, the administration of the TSPO ligand, YL-IPA08, to rats subjected to chronic unpredictable stress (CUS) increased progesterone and Allo levels in the hippo- campus and prefrontal cortex and induce antidepressant effects [221]. Moreover, the TSPO activation and the subsequent neuro- steroid-induced synthesis contribute to maintain hippocampal morphologic and functional plasticity and to prevent the dys- function of HPA axis observed in CUS rats [221]. After the admin- istration of another TSPO ligand, etifoxine, rats showed a reduction of anxiety-like behavior, which correlated with increased Allo concentrations [222]. The administration of the 5 α-reductase inhibitor, finasteride blocked etifoxine’s pharmaco- logical and behavioral effects, confirming that etifoxine acts by inducing neurosteroidogenesis [217]. Clinical studies have pro- ven that etifoxine can improve anxiety-related disorders in humans [223]. Thus, TSPO ligands and other drugs that promote neurosteroidogenesis and increase Allo concentrations are pro- mising therapeutic strategies for MDD and PTSD.

2.6.2. The endocannabinoid system

The endocannabinoid system is involved in HPA axis activation and its interaction with glucocorticoids is useful to cope with stress. Furthermore, the endocannabinoids modulate fear mem- ory and other memory processes, like reconsolidation and extinction, which are a core feature of PTSD [224]. In a mouse model of depression, the hippocampal suppression of the endocannabinoid signaling leads to depressive-like behavior [225,226], thus suggesting it may play a role in PTSD and MDD.

The endocannabinoid receptor type 1 (CB1) has received growing attention in mood disorders. A positron emission tomography study showed enhanced expression in individuals with PTSD but not in trauma-exposed healthy controls [227].

Furthermore, this study found diminished peripheral level of the endocannabinoid, anandamide (AEA), which suggests that the enhanced CB1 receptor expression, and probably sensitivity, may be in part due to lower levels of AEA [227]. Likewise, a postmortem study revealed higher expression of CB ₁ in depressed suicide victims [228]. However, genetic studies

support the hypothesis that impairment in CB ₁ may increase the risk to develop depression and other psychiatric pathologies.

SNPs in the CB 1 gene can increase the vulnerability to develop depressive episode after trauma exposure and in patients with mood disorders when the frequency of SNPs increases [229,230].

Moreover, AEA and the congener, 2-arachidonoylglycerol (2-AG) serum concentration is lower in the plasma of depressed women than in matched control subjects [231]. The levels of these endocannabinoids in depressed women are a focus of investiga- tion and several considerations of whether they may be valuable markers are being discussed. Likewise, rodent models of PTSD and depression show decreased levels of AEA and 2-AG [232]. For example, in CUS rats, the number of CB1 receptor binding sites increase, while the levels of AEA decrease in the prefrontal cortex, ventral striatum, and hippocampus [233]. The administra- tion of AM404, an endocannabinoid reuptake blocker, facilitates fear extinction in rats by enhancing AEA and 2-AG concentra- tions [234]. This evidence confirms the relevance of AEA levels to facilitate fear extinction in the basolateral amygdala [235]. In human studies, AEA concentrations are reduced in PTSD patients compared both to healthy controls and to trauma-exposed con- trols [227].

Other endocannabinoid-like molecules that activate the peroxisome proliferator-activated receptor-alpha (PPAR- α),

such as N-oleoyld-opamine (OEA) and

N-palmitoylethanolamine (PEA) may be involved in the patho- physiology of PTSD and MDD [236]. PPAR- α activation has been shown to mediate the response to stressful conditions [237]. In healthy adults, PEA significantly increases in clinical stress tests in connection with an increase of cortisol levels [238]. PEA levels also decrease when healthy subjects experi- ence a short-term depressed mood [239]. On the other hand, PEA levels in PTSD, MDD, and impulsive aggression are decreased [240]. Of note, PEA adjunctive therapy to citalopram improves depressed symptoms [241] and intense physical activity increases PEA and OEA levels while improving depres- sion and PTSD symptoms [242]. The relationship between PPAR- α and emotional regulation is further highlighted by its role as an anti-neuroinflammatory target [243 – 247]. In recent studies originated in our laboratory we have shown that stimulation of PPAR- α by PEA or synthetic agonists increased corticolimbic Allo levels in hippocampus, amygdala, and prefrontal cortex, which facilitated contextual fear extinction and fear extinction retention, and improved aggression and anxiety-like behavior in SI mice [193,248].

PEA also induces antidepressant-like effects in SI mice [193].

Furthermore, this behavioral improvement by PEA or other PPAR- α synthetic agonists, which was associated with normal- ization of Allo levels, was blunted by antagonism at PPAR- α, inhibition of Allo biosynthetic enzymes, and in PPAR- α KO mice. Other rodent studies have reported that exposure to predator stressors downregulates PEA and OEA levels [249], but treatments that restore PEA and OEA levels result in anti- depressant-like effects [250 – 252].

AEA and other CB1 endogenous agonists are of interest as

biomarkers in PTSD and MDD diagnose and possibly as end-

points of treatment outcome. Furthermore, preclinical and

clinical studies support an emerging role of PPAR- α in MDD

and PTSD. The discovery of this new interrelation between

PPAR- α activation and Allo biosynthesis may unveil a biomarker axis uniquely altered at the interface of the endo- cannabinoid and the neurosteroid systems (Figure 3).

3. Blood-based biomarker axis

3.1. The potential of peripheral biomarkers

All biomarkers discussed in this review article have been studied in animal models or in humans, with a focus on the CNS and CSF, serum, and plasma [253,254]. Noninvasive per- ipheral biomarkers are more useful and functional for diag- nosis of disorders of mood and emotions. CSF reflects more closely the alterations of the brain, but the procedure is stressful and results in pain and discomfort in the patients [255,256]. Blood draws can also be stressful for some patients, putting at risk a precise diagnosis or may even turn patients away from testing. However, most studies are currently assessing serum- or plasma-based diagnostic tests [257], considering different biomarkers and several methods, including metabolomics (neurohormones, neuroactive ster- oids, endocannabinoids) or genomics and proteomics. The majority of techniques used for proteomic analyses are based on the combination of two-dimensional gel electrophoresis (2DE) to separate proteins and mass spectrometry (MS) for their identification. The main advantage of this technique consists in the quantification of several biomarkers simulta- neously in small sample amounts; while, the disadvantages include very low reproducibility and the need of targeted analyses to discover a potential biomarker. Despite the power to separate several proteins simultaneously, the 2DE- MS technique is limited by the difficulty in detecting low- abundance proteins and proteins with extremely high or low molecular weight [258]. An alternative MS-based approach is shotgun proteomics or liquid chromatography-tandem mass spectrometry (LC-MS/MS), which provides several chromato- graphic steps prior to MS analyses. Shotgun proteomics has the advantage of being more sensitive in detecting differ- ences in proteins expressed in patients with MDD [259], as well as alterations in protein expression in patients with schizophrenia, which were not detected by using 2DE-MS [260]. Validation of the potential biomarkers are additionally assessed by employing other methods, such as Western blot, or the enzyme-linked immune-adsorbent assay (ELISA) [261].

Another valuable proteomic method, which allows to simultaneously measuring multiple proteins in a small volume of sample, is the Luminex bead-based approach. This techni- que has been successfully used for biomarker discovery and is more common in several clinical studies such as in studies of autoimmune disorders [262], or Alzheimer ’s disease [ 263].

Several other techniques involve the use of stable isotopes to label peptides. The most widely known is the Isotope Coded Affinity Tags, which permits to distinguish between different protein in one sample based on their isotopic com- position, separating the peptide fragments of interest and quantifying them by MS [264]. By labeling only cysteine resi- dues, this approach only analyzes proteins containing this amino-acid residue, hence excluding many other relevant proteins.

More recently, the development of the isobar tags for relative and absolute quantification (iTRAQ) technology improved the quality of protein identification and quantifica- tion, reducing the assay variations observed in multiple MS quantification [265]. The combined use of iTRAQ and LC-MS /MS is a promising technology for protein identification [266].

The iTRAQ-LC-MS/MS technology is one of the most sensitive proteomic assays that can quantitatively analyze low- abundance proteins in biological samples [267]. This approach has been employed in different disorders, including cardiovas- cular disease, neurological, and psychiatric disorders [266,268,269]. Interestingly, the iTRAQ approach enables the identification of human peripheral serum protein expression that may offer relevant biomarkers for the diagnosis of depres- sion [270] and allows distinguishing between MDD and other psychiatric disorders, such as bipolar disorder [271]. The iTRAQ methodology detected differences in serum expression between depressed patients and healthy controls and ELISA confirmed a significant increase of CRP, ITIH4, SAA1, and ANGPTL3 protein levels in patients with clinical depression compared to healthy controls. Thus, these encouraging results suggest that iTRAQ is a useful methodological tool to identify candidate biomarkers in serum from patients. For the routine evaluation of protein levels, ELISA tests are more practical and time effective.

In our laboratory, with the goal of assessing biomarkers, we employ the gas chromatography-mass spectrometry (GC-MS) to determine neuroactive steroids in CSF, serum, and plasma of MDD and PTSD patients but also in serum and discrete brain regions of mouse models of these disorders. Our results have identified specific changes in the axis of neuroactive steroid biosynthesis and their relation with neurotransmitter systems, including GABA A receptors [193] (Figure 3 and Figure 5). While the GC-MS technique offers a powerful tool in biomarker discov- ery, the feasibility of using the GC-MS technology as a routine test to evaluate biomarker concentrations in tissue of patients and individuals at risk is limited by its costs, complexity and it is time-consuming. However, once firmly established and structure selectivity achieved, the biomarkers ’ evaluation may be substi- tuted, in a later stage, by ELISA determinations to satisfy the market time and cost requirements.

In addition, gene-activity assay is a promising technique and cost-efficient; however, this technology requires more investiga- tion to identify specific genes that change their expression in PTSD and MDD [88]. DNA microarrays can efficiently highlight gene expression profiling of transcriptional reactivity. Currently, studies that analyze gene expression related to PTSD showed relevant signatures in mononuclear cells that may be useful to diagnose a mental disorder [272]. However, improved methods are still required to screen more efficiently through sets of candidate variants, and then, a rigorous validation of variants and gene effects are also needed [273]. Furthermore, replications in larger samples and investigations focusing on selected markers as part of the biosignatures that have been discovered, are required to assess the diagnostic utility and pathological relevance of these methods.

Several experimental protocols that consider not one or a

few, but several biomarkers are advantageous and have been

recently proposed [261,274]. These protocols provide the eva-

luation of many biomarkers together. For example, Papakostas